Expression system for psicose epimerase and production for psicose using the same

ABSTRACT

A gene expression cassette capable of producing psicose at high yield with high stability, a GRAS (Generally recognized as safe) microorganism, a method of producing the enzyme by using the GRAS microorganism, and a method of producing the psicose by using the GRAS microorganism and enzyme are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean PatentApplication No. 10-2014-0149019 filed Oct. 30, 2014, on and KoreanPatent Application No. 10-2015-0072090 filed in the Korea IntellectualProperty Office on May 22, 2012, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention an expression system being capable of producingPsicose epimerase having a high conversion rate and stability, generallyrecognized as safe (GRAS) microorganism including the expression system,and a method of producing psicose by using the microorganism and theenzyme.

BACKGROUND ART

Various biosynthetic compounds are produced in the natural metabolicprocess and used in different industrial fields such as food, feedstuff,cosmetic, and pharmaceuticals. The compounds have been produced by usingbacterium or other microorganism developed for producing and secretingthem in a large scale. For examples, Corynebacterium species has beenused in the industry of amino acid production. In middle of 1950,Corynebacterium glutamicum producing glutamic acid efficiently and,auxotrophic mutant of Corynebacterium glutamicum has produced variousamino acids by using fermentation.

The expression of various related genes can be regulated accurately forcell engineering therefore requiring the efficient expression system.Different components of cell regulating sequence have been known in theart. The examples of the components are a binding region to theoperator, a RNA polymerase binding region of −35 and −10 regions, and aribosome binding site or Shine-Dalgarno sequence in ribosomal 16S RNA.

It is important to select a promoter to develop the expression system,because the promoter is largely involved in the gene expression leveland expression regulation. Several promoters being applicable toCorynebacterium glutamicum have been reported and are derived fromCorynebacterium sp. or E. coli (J. Biotechnol., 104:311-323, 2003).

However, the promoter derived from E. coli a low permeability of anexpression inducer and absence of gene expression inhibitor and thusshows low activity relative to that of Corynebacterium. Even if the samepromoters are used, their activities are different depending on thecoating sequence of target gene. The promoters used in Corynebacteriumhave a difficulty in being prepared for the desired object, because itis a narrow of choice in the expression level of promoters. Especially,when the expressions of various genes are regulated together such as theestablishment of metabolic pathway, Corynebacterium, various promoterscannot be selected, unlike E. coli.

Psicose is getting a spotlight in a diet sweetener, but is required tobe produced in a large scale for applying to the food due to rare sugarin nature. In the prior art, Psicose has been largely produced bysynthetic chemical method. As the enzymatic method, KR10-0744479discloses the mass production of psicose using the psicose epimeraseproduced by E. coli transformed with the coding gene of psicoseepimerase derived from Agrobacterium tumefaciens. There is a method ofproducing psicose by using a microorganism producing enzyme withoutpurification in a low production cost. In the disclosure ofKR10-1106253, the recombinant E. coli which is transformed with thecoding gene of psicose-3-epimerase derived from Agrobacteriumtumefaciens and includes an inactivated specific gene, is inoculated onthe culture medium including fructose to convert the fructose topsicose.

The recombinant E. coli used in KR10-1106253 is not GRAS (GenerallyRecognized As Safe) microorganism, and thus cannot be suitable in foodindustry. In addition, the Psicose epimerase derived from Agrobacteriumtumefaciens has a low enzyme activity and heat stability.

Therefore, there is a need to develop an expressing being capable ofproducing the Psicose epimerase having a high enzyme activity in GRASmicroorganism at a high yield and stably expression system, a method ofpsicose epimerase using the expression system, and a method of psicoseby using the enzyme or transformed GRAS microorganism.

DISCLOSURE Technical Problem

An embodiment provides a promoter being capable of producing psicose athigh yield with high stability.

Another embodiment provides a regulating sequence being capable ofregulating expression of Psicose epimerase in Corynebacterium sp. andcontaining the promoter.

A further embodiment provides a gene expression cassette including anucleotide sequence encoding the psicose epimerase and the promoter orthe regulating sequence.

A still further embodiment provides a vector being used inCorynebacterium sp., including a gene expression cassette including anucleotide sequence encoding the psicose epimerase and the promoter orthe regulating sequence.

Still another embodiment provides a Corynebacterium sp. cell expressingthe psicose epimerase, including the gene expression cassette or beingtransformed by the gene expression cassette.

Still another embodiment provides a composition for the production ofpsicose, comprising at least one selected from the group consisting ofPsicose epimerase, a recombinant cell, a culture of the recombinantcell, a lysate of the recombinant cell and an extract of culture.

Yet another embodiment provides a method of producing psicose, using atleast one selected from the group consisting of Psicose epimerase, arecombinant cell, a culture of the recombinant cell, a lysate of therecombinant cell and an extract of culture.

Technical Solution

The present invention relates to a gene expression cassette capable ofproducing psicose at high yield with high stability, a GRAS (Generallyrecognized as safe) microorganism, a method of producing the enzyme byusing the GRAS microorganism, and a method of producing the psicose byusing the GRAS microorganism and enzyme.

The promoter derived from E. coli shows a low activity inCorynebacterium sp., because the expression inducing factor has a lowpermeability, and the gene expression material does not exist inCorynebacterium sp. Therefore, the present invention can provide apromoter being suitable for expressing a Psicose epimerase inCorynebacterium sp.

The present invention provides a promoter being capable of producingpsicose at high yield with high stability, a regulating sequenceincluding the promoter, and a gene expression cassette including theregulating sequence, thereby producing psicose epimerase inCorynebacterium sp. at a high yield with high stability in a largeamount. In addition, the present inventors provide a psicose epimerasewhich can be expressed at a high rate in combination with the promoter,and a nucleotide sequence encoding the psicose epimerase.

Herein, the term “promoter”, “nucleotide molecule having a promoteractivity” or “promoter sequence” means a nucleotide molecule beingcapable of regulating the transcription or the expression of anucleotide of interest, with being operably connected to the nucleotideof interest. The promoter can include a transcription promoter andexpression promoter.

The nucleotide sequence of interest may not be linked to the promoterchemically, and can be linked to the promoter by using additional generegulating sequence and/or linker nucleotide sequence, and the like.Preferably, the nucleotide sequence of interest to be transcribed can belocated at a downstream of promoter (i.e., 3′-end of promoter sequence).The interval between the promoter sequence and the nucleotide sequenceto be transcribed can be preferably 200 base pairs or less, or morepreferably 100 bp or less.

Herein, the term “ribosome binding site” (RBS) or “Shine-Dalganosequence” means a region of A/G rich polynucleotide sequence which isbound by a ribosome for translation.

Herein, the term “regulating sequence” means a nucleotide moleculehaving a regulating activity of gene expression such as transcriptionand/or translation of target polynucleotide and being operably linked tothe target polynucleotide. The regulating sequence may be called as“regulating nucleotide sequence.”

Hereinafter, the term “expression cassette” includes a regulatingsequence being operably linked to the target nucleotide sequence, suchas a nucleotide sequence coding the Psicose epimerase. Therefore, theexpression cassette may include a nucleotide sequence required forexpressing a protein after the transcription or the translation, as wellas a nucleotide sequence regulating the transcription or thetranslation.

In the present invention, the nucleotide molecule is preferably anon-naturally occurring molecule, an isolated molecule or, a syntheticor recombinant type. The term, “isolated” nucleotide molecule may notinclude other nucleotide molecule in natural source, other cellularmaterial or any component of culture medium in case of the recombinationproduction method, or other chemical precursor or other chemicals incase of the chemical synthesis method.

In an embodiment, the regulating sequence used for Corynebacterium sp.can express the psicose epimerase having a high stability and enzymeactivity in GRAS microorganism at a high yield and stability.

The present invention is described in detail hereinafter.

An embodiment of present invention provides a regulating sequence beingoperated in Corynebacterium sp. and thus a psicose epimerase having ahigh stability and activity can expressed in GRAS microorganism.

Another embodiment provides a gene expression cassette, producing apsicose epimerase in Corynebacterium sp., and comprising a nucleotidesequence encoding the psicose epimerase; and a regulating sequence beingoperably connected to the nucleotide sequence in the upstream andregulating the expression of the nucleotide sequence in Corynebacteriumsp, wherein the regulating sequence comprising a promoter including anucleotide sequence of SEQ ID NO: 1.

The regulating sequence includes a nucleotide sequence encoding thepsicose epimerase; and a promoter expressing the psicose epimerase inGRAS microorganism, for example Corynebacterium sp. or a regulatingsequence including the promoter. The regulating sequence can be anunmodified or modified nucleotide sequence which regulate the expressionof nucleotide sequence encoding the Psicose epimerase in Corynebacteriumsp.

The promoter includes a nucleotide molecule of SEQ ID NO: 1 andfunctional variants thereof. In an embodiment, the functional variant ofpromoter, shows at least 90% nucleotide sequence identity, for examples,at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%.

The gene expression cassette or the regulating sequence furthercomprises at least one sequence selected from the group consisting of aribosome binding site (RBS) sequence, a spacer sequence and a linkersequence.

The ribosome binding site sequence can be included at least one or morecopy, for examples 1 to 5 copies, or 2 copies in the regulatingsequence.

The regulating sequence may include a first RBS sequence and a firstspacer sequence; a first RBS sequence, a first spacer sequence and asecond RBS sequence which is connected to 3′-end of the first spacerdirectly or via a linker; and a first spacer sequence and a second RBSsequence which is connected to 3′-end of the first spacer directly orvia a linker; and a second spacer.

Specifically, the regulating sequence includes one copy of RBS sequence.For example the regulating sequence includes a promoter of SEQ ID NO: 1,a first RBS sequence of SEQ ID NO: 2 and a first spacer selected fromthe group consisting of the sequences of SEQ ID NO: 3 to SEQ ID NO: 6(promoter-RBS1-Spacer1). Optionally, the regulating sequence furtherincludes a linker sequence in size of 1 to 100 bp which is connected to3′ end of the first spacer (promoter-RBS1-Spacer1-linker).

The regulating sequence includes two copies of RBS sequence. For examplethe regulating sequence includes (i) a promoter of SEQ ID NO: 1, andfurther includes at least one selected from the group consisting of (ii)a first RBS sequence of SEQ ID NO: 2, (iii) a first spacer selected fromthe group consisting of the sequences of SEQ ID NO: 3 to SEQ ID NO: 6,(iv) a linker sequence of SEQ ID NO: 12, and (v) a second spacerselected from the group consisting of the sequences of SEQ ID NO: 7 to11. For examples, the second RBS sequence may be connected to 3′-end ofthe first spacer directly or via a linker (promoter-RBS1-Spacer1-RBS2,or promoter-RBS1-Spacer1-linker-RBS2). In the regulating sequenceincluding two copies of RBS sequence, a second spacer can be connectedto 3′-end of the second RBS sequence. In addition, the combination offirst RBA and first spacer, or second RBS and second spacer can berepeated at least one or more, for examples 1 to 5 times, 2 times, 3times, 4 times or 5 times.

The specific examples of unmodified regulating sequence are shown in thefollowing.

(1) a promoter and a combination of RBS and space sequence which arelinked to 3′-end of the promoter directly or via a linker (e.g.,promoter-linker-RBS1-Spacer1, or promoter-RBS1-Spacer1),

(2) a promoter and at least two combinations of RBS and space sequence(e.g., promoter-RBS1-Spacer1-RBS2-Spacer2), and

(3) a promoter, at least two combinations of RBS and space sequence, anda linker which is located between the first spacer sequence and thesecond RBS (e.g., promoter-RBS1-Spacer1-linker sequence-RBS2-Spacer2).

In an embodiment, the ribosome binding site sequence is a nucleotidesequence in a size of 7 to 20 bp which including a nucleotide sequenceof SEQ ID NO: 2, for example a nucleotide sequence of SEQ ID NO: 2.

The linker sequence is a nucleotide sequence in a size of 5 to 100 bp,or 5 to 80 bp, for example a nucleotide sequence of SEQ ID NO: 12.

The spacer sequence in the regulating sequence may be in the length of 3to 15 bases of various bases, and increase the expression efficiency ofgene located in downstream of the regulating sequence. The spacersequence can be prepared in various base composition and base length byconsidering the gene of interest, the kind of host cell, and the like.

The modified regulating sequence of the present invention includes atleast one base which substitutes at least one base of at least oneselected from a first spacer and a second spacer.

For example, when the modified regulating sequence includes one copy ofRBS, the modified regulating sequence includes promoter, first RBS andfirst spacer of which TT of a first base and a second base can besubstituted with GA, GT or GC base.

When the modified regulating sequence includes two copies of RBS, TT ofa first base and a second base of the first spacer connected to 3′-endof the first RBS can be substituted with GA, GT or GC; TT of a firstbase and a second base of the second spacer connected to 3′-end of thesecond RBS can be substituted with GG, GA, GT or GC; or TT of a firstbase and a second base of the first spacer can be substituted with GA,GT or GC and TT of a first base and a second base of the second spacercan be substituted with GG, GA, GT or GC.

For example, the first spacer sequence can be at least a nucleotidesequence selected from the group consisting of nucleotides of SEQ ID NO:3 to 6. The second spacer sequence can be at least a nucleotide sequenceselected from the group consisting of nucleotides of SEQ ID NO: 7 to 11.

In an embodiment, the promoter sequence, the RBS sequence, the firstspacer and the second spacer, and their modified sequence, and thelinker being applicable to the regulating sequence are exemplified inTable 1.

TABLE 1  Seq ID No sequence(5′ -> 3′) name  1aagcgcctcatcagcggtaaccatcacggg promoter ttcgggtgcgaaaaaccatgccataacaggaatgttcctttcgaaaattgaggaagcctt atgcccttcaaccctacttagctgccaattattccgggcttgtgacccgctacccgataa ataggtcggctgaaaaatttcgttgcaatatcaacaaaaaggcctatcattgggaggtgt cgcaccaagtacttttgcgaagcgccatctgacggattttcaaaagatgtatatgctcgg tgcggaaacctac  2 gaaagga RBS  3ttttttaccc 1^(ST) SPACER R1TT  4 gattttaccc 1^(ST) SPACER R1GA  5gtttttaccc 1^(ST) SPACER R1GT  6 gcttttaccc 1^(ST) SPACER R1GC  7ttacaaa 2nd SPACER R2TT  8 gaacaaa 2nd SPACER R2GA  9 gtacaaa 2nd SPACERR2GT 10 gcacaaa 2nd SPACER R2GC 11 ggacaaa 2nd SPACER R2GG 12atggctgtatacgaactcccagaactcga linker ctacgcatacgac

The regulating sequence includes at least a polynucleotide selected fromthe group consisting of the sequences shown in SEQ ID NO: 13 to SEQ IDNO: 32, and regulates the expression of psciose epimerase inCorynebacterium sp.

TABLE 2 SEQ ID NO sequence(5′ -> 3′) name 13 (SEQ ID NO: 1) +gaaagga ttttttaccc RBS1/1^(st) SPACER-TT 14 (SEQ ID NO: 1) +gaaagga gattttaccc RBS1/1^(st) SPACER-GA 15 (SEQ ID NO: 1) +gaaagga gtttttaccc RBS1/1^(st) SPACER-GT 16 (

1) + gaaagga gcttttaccc RBS1/1^(st) SPACER-GC 17 (SEQ ID NO: 1) +gaaagga ttttttaccc RBS1/1^(st) SPACER-TTatggctgtatacgaactcccagaactcgactacgcatacgac linker 18 (SEQ ID NO: 1) +gaaagga gattttaccc SOD-R1GA/R2TTatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga ttacaaa 19(SEQ ID NO: 1) + gaaagga gattttaccc SOD-R1GA/R2GAatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gaacaaa 20(SEQ ID NO: 1) + gaaagga gattttaccc SOD-R1GA/R2GTatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gtacaaa 21(SEQ ID NO: 1) + gaaagga gattttaccc SOD-R1GA/R2GCatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gcacaaa 22(SEQ ID NO: 1) + gaaagga gattttaccc SOD-R1GA/R2GGatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga ggacaaa 23(SEQ ID NO: 1) + gaaagga gtttttaccc SOD-R1GT/R2TTatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga ttacaaa 24(SEQ ID NO: 1) + gaaagga gtttttaccc SOD-R1GT/R2GAatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gaacaaa 25(SEQ ID NO: 1) + gaaagga gtttttaccc SOD-R1GT/R2GTatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gtacaaa 26(SEQ ID NO: 1) + gaaagga gtttttaccc SOD-R1GT/R2GCatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gcacaaa 27(SEQ ID NO: 1) + gaaagga gtttttaccc SOD-R1GT/R2GGatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga ggacaaa 28(SEQ ID NO:1) + gaaagga gcttttaccc SOD-R1GC/R2TTatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga ttacaaa 29(SEQ ID NO: 1) + gaaagga gcttttaccc SOD-R1GC/R2GAatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gaacaaa 30(SEQ ID NO: 1) + gaaagga gcttttaccc SOD-R1GC/R2GTatggctgtatacgaactcccagaactcgactacgcatacgac gaaagga gtacaaa 31(SEQ ID NO: 1) + gaaagga gcttttaccc SOD-R1GC/R2GCatggctg tatacgaact cccagaactc gactacgcat acgac gaaagga gcacaaa 32(SEQ ID NO: 1) + gaaagga gcttttaccc SOD-R1GC/R2GGatggctg tatacgaact cccagaactc gactacgcat acgac gaaagga ggacaaa

The regulating sequence of the present invention can regulate theexpression of psicose epimerase connected to the regulating sequence inthe downstream in Corynebacterium sp. Therefore, the gene expressioncassette of the present invention can be used for expressing the targetgene in Corynebacterium sp., and the target gene can be a nucleotidesequence encoding psicose epimerase. The psicose epimerase can bederived from Clostridiun scidens, Treponema primitia, or Ensiferadhaerens. The psicose epimerase derived from Agrobacterium tumefacienshas a low enzyme activity and heat stability, and thus is notpreferably.

The promoter derived from E. coli shows a low activity inCorynebacterium sp., because the expression inducing factor has a lowpermeability, and the gene expression material does not exist inCorynebacterium sp. Even though the same promoter is used, the promoterexpression activity can be varied depending on the target gene to beexpressed. The promoter being applicable to Corynebacterium sp. showslow promoter activity and Corynebacterium sp. dose not provide a widechoice of promoter. Therefore, the promoter being suitable forCorynebacterium sp. cannot be prepared easily. Although the promoter issuitably used in Corynebacterium sp., the promoter may have differentregulating acidity of transcription or expression depending on the kindof target gene. The promoter, the regulating sequence and the geneexpression cassette of present invention are very preferably used forexpressing psciose epimerase in Corynebacterium sp.

The coding sequence of target protein may be connected to 3′-end of theregulating sequence used in Corynebacterium sp. directly or via alinker.

The psicose epimerase having a high enzyme activity and heat stabilityis preferably used. It is important to combine the promoter or theregulating sequence with the coding sequence of psicose epimerase. Thecoding sequence of psicose epimerase can provide a preferable expressionlevel of protein, when it is used with the promoter of presentinvention, and a high heat stability can be obtained due to the goodprotein folding. The coding sequence of psicose epimerase according tothe present invention is preferable to be used together with thepromoter or the regulating sequence of the present invention.

In an embodiment, the psicose epimerase is derived from Clostridiunscidens, Treponema primitia, Ensifer adhaerens or Ruminococcus torques,and preferably at least an amino acid sequence shown in SEQ ID NO: 33 to36.

As long as the psicose epimerase maintains the enzyme activity ofconverting fructose to psicose, any modified sequence of amino acidsequence shown in SEQ ID NO: 33 to 36 can be used by obtainingsubstitution, insertion and/or deletion of the partial amino acid. Forexample, the modified sequence can include an amino acid sequence havingan amino acid sequence identify of 70% or higher, 80% or higher, 90% orhigher, 95% or higher, or

99% or higher, compared to the amino acid sequence shown in SEQ ID NO:33 to 36.

The coding sequence of psicose epimerase can be a nucleotide sequence ofpsicose epimerase derived from Clostridiun scidens, Treponema primitia,Ensifer adhaerens or Ruminococcus torques, or a modified sequencedobtained by optimizing the coding sequence to be suitable for expressionin E. coli or Corynebacterium sp.

For example, the nucleotide sequence encoding the psicose epimerase canbe a coding sequence of any one amino acid sequence selected from thesequences of SEQ ID NO: 33 to 36. Specifically, the nucleotide sequencecan be any one selected from the sequences of SEQ ID NO: 37 to SEQ IDNO: 44, or a nucleotide sequence having substantially the same sequencehomology to them. The term, substantially the same sequence homologymeans that any nucleotide sequence have the nucleotide sequence identityof 70% or higher, 90% or higher, or 98% or higher, compared to at leasta nucleotide sequence selected from SEQ ID NO: 37 to SEQ ID NO: 44, whenany nucleotide sequence is aligned with the nucleotide sequence selectedfrom the sequences of SEQ ID NO: 37 to SEQ ID NO: 44 and is performed tosequence analysis.

In an embodiment, the psicose epimerase derived from Clostridiun scidens(CDPE) includes an amino acid sequence of SEQ ID NO: 33, and anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33,for example the nucleotide sequence of SEQ ID NO: 37 or SEQ ID NO: 38

In an embodiment, the psicose epimerase derived from Treponema primitia(TDPE) includes an amino acid sequence of SEQ ID NO: 34, and anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34,for example the nucleotide sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

In an embodiment, the psicose epimerase derived from Ensifer adhaerens(EDPE) includes an amino acid sequence of SEQ ID NO: 35, and anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35,for example the nucleotide sequence of SEQ ID NO: 41 or SEQ ID NO: 42.

In an embodiment, the psicose epimerase derived from Ruminococcustorques (RDPE) includes an amino acid sequence of SEQ ID NO: 36, and anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36,for example the nucleotide sequence of SEQ ID NO: 43 or SEQ ID NO: 44.

The gene expression cassette of the present invention may comprisefurther at least a sequence selected from the group consisting of areplication origin, leader sequence, a selection marker, a cloning site,and a restriction enzyme recognition site.

In another embodiment, the gene expression cassette useful forCorynebacterium sp. comprising a promoter used for Corynebacterium sp.,a regulating sequence including the promoter, or the regulating sequenceand a polynucleotide encoding the psicose epimerase, Corynebacterium sp.expression cassette is provided.

The promoter used for Corynebacterium sp., the regulating sequence andthe psicose epimerase are described in the above.

The gene expression cassette of the present invention may comprisefurther at least a sequence selected from the group consisting of areplication origin, leader sequence, a selection marker, a cloning site,and a restriction enzyme recognition site.

The gene expression cassette may be used in a naked polynucleotideconstruct, or in a recombinant vector. The term, a recombinant vectormeans a nucleotide molecule being capable of transferring a targetpolynucleotide which is operably linked to the recombinant vector. Thetarget polynucleotide can be operably connected to a transcriptionregulator such as the promoter and a transcription terminator.

The recombinant vector can be a cloning vector or an expression vectoraccording to the method known widely in the art (Francois Baneyx,current Opinion Biotechnology 1999, 10:411-421). The recombinant vectormay be any vector which has been used for genetic recombination, and beany one selected from plasmid vector and viral vector (e.g.,replication-deficient retrovirus, adenovirus and adenovirus associatedvirus)

viral vector having an equivalent activity to the vector. The examplesof recombinant vectors include at least a vector selected from the groupconsisting of pET, pKK223-3, pTrc99a, pKD, pXMJ19, pCES208 vector, andthe like. Preferably, the vector may be E. coli-Corynebacterium shuttlevector (pCES208, J. Microbiol. Biotechnol., 18:639-647, 2008).

Accordingly, the vector including the gene expression cassette can be anexpression vector such as a plasmid which can grow in Corynebacteriumsp. and express the target protein.

The transcription terminator can be rrnB, rrnB_T1, rrnB_T2, or T7terminator, or preferably T7 terminator derived from pET21a vector.

In an embodiment, a vector includes a promoter having a nucleotidesequence shown in SEQ ID NO: 1, or only a regulating sequence withoutthe target gene. The vector can be a shuttle vector, a replicationvector or an expression vector which can grow in E. coli andCorynebacterium sp.

In particular, the present invention provides a vector such as a plasmidincluding a regulating sequence to regulating the expression targetpolynucleotide sequence with being located in the upstream of targetpolynucleotide sequence. The regulating sequence may include a promoterhaving a nucleotide sequence of SEQ ID NO: 1, a first ribosome bindingsite (RBS) sequence and a first spacer sequence.

The regulating sequence may include a promoter, a first RBS sequence, afirst spacer sequence, and a second RBS sequence connected to 3′-end ofthe first spacer directly or via a linker. The regulating sequenceincludes a promoter, a first RBS sequence, a first spacer sequence, asecond RBS sequence and a second spacer sequence connected to 3′-end ofthe second RBS directly or via a linker.

In the vector including the promoter, the first spacer sequencecomprises a modified nucleotide sequence where first base and secondbase (TT) in the nucleotide sequence of SEQ ID NO: 3 are substitutedwith GA, GT or GC. The modified first spacer can include a sequence ofSEQ ID NO: 4, 5, or 6.

When the vector includes the first spacer and the second spacer, eitheror both of the first spacer and the second spacer can include at least amodified base. For example, the first spacer sequence includes aunmodified nucleotide sequence of SEQ ID NO: 3, but the second spacersequence includes a modified nucleotide sequence a modified nucleotidesequence where first and second base in the nucleotide sequence of SEQID NO:7 are substituted with GA, GT, GC or GG. The modified nucleotidesequence of second spacer may include a nucleotide sequence selectedfrom the sequences of SEQ ID NO: 8 to 11.

Alternatively, the first and second base (TT) of the first spacersequence having a nucleotide sequence of SEQ ID NO: 3 may be substitutedwith GA, GT or GC, and, the first and second base (TT) of the secondspacer sequence having a nucleotide sequence of SEQ ID NO: 7 may be TT,GA, GT, GC or GG. The examples of first spacer sequences are anucleotide sequence of SEQ ID NO: 4 to 6 and the examples of secondspacer sequence are a nucleotide sequence of SEQ ID NO: 7 to 11.

In the vector including only a regulating sequence without a targetgene, the expression cassette and the vector including the expressioncassette including RBS, linker, first spacer, second spacer, a codingsequence of psicose epimerase and a regulating sequence are the same asdescribed in the above.

In an embodiment, a recombinant Corynebacterium sp. cell including thegene expression cassette or transformed by the expression cassette canbe provided.

The method of transforming a host cell by the recombinant vector can beperformed by any transforming method which has been known to anordinarily-skilled person in the art without limitation. For example,illustrative, non-limiting examples of the method include protoplastfusion, electroporation, projectile bombardment, and infection with aviral vector.

The transformed Corynebacterium sp. of present invention shows a highstability and expression efficiency of introduced psicose epimerase, andthus can maintains the high conversion rate of psicose for a long time.The transformed Corynebacterium sp. can be applied usefully to theproduction of psicose and increase the production yield of psicose.

Preferred Corynebacterium sp. may be Corynebacterium glutamicum,Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum,Corynebacterium thermoaminogenes, Corynebacterium melassecola orCorynebacterium efficiens.

The transformed Corynebacterium sp. of present invention may be arecombinant Corynebacterium glutamicum.

The culture of Corynebacterium sp. can be performed in the suitablemedium according to the known method in the art. The culturing of therecombinant cell may be conducted under a medium and condition readilyselected according to the property of the strain (host cell) by thoseskilled in the art. For example, the culturing may be a continuous-typeculture, a semi-continuous-type culture, or a batch-type culture, but isnot limited thereto. The culture medium being applicable to the presentinvention includes carbon source, nitrogen source, inorganic salts,vitamin and/or trace element. The preferred carbon sources includesaccharide such as monosaccharide, disaccharide or polysaccharide. Tomaintain the metal ion concentration, the chelating agent can be addedto the culture medium. All components of culture medium can besterilized by heating at 1.5 bar and 121° C. for 20 minutes, orsterilization filtering.

In an embodiment, a composition for production of psicose including atleast one selected from the group consisting of an psicose epimeraseobtained by using the recombinant Corynebacterium sp., a recombinantcell, a culture of the recombinant cell, a lysate of the recombinantcell and an extract of cell culture can be provided.

In another embodiment, a method for producing psicose including a stepof reacting fructose-containing substrate with a composition forproduction of psicose including at least one selected from the groupconsisting of an psicose epimerase obtained by using the recombinantCorynebacterium sp., a recombinant cell, a culture of the recombinantcell, a lysate of the recombinant cell and an extract of cell culture orcell lysate can be provided.

The culture can contains an enzyme protein produced from the recombinantCorynebacterium sp cell, and may include the recombinant cell, or mayalternatively be in a cell-free form. The lysate may result from thelysis of the recombinant cell or may include a supernatant obtained bycentrifuging the lysate, so that it contains the enzymatic proteinproduced from the recombinant cell in n either case. Unless statedotherwise herein, the recombinant cell means at least one selected fromthe group consisting of a cell mass of the strain, a culture of thestrain and a lysate of the strain.

The method of producing psicose includes a step of reacting theCorynebacterium sp. with fructose-containing substrate. In oneembodiment, the reaction between the enzymatic proteins and fructose maybe carried out by culturing a cell mass of the recombinant cell in amedium containing fructose. The reaction of the Corynebacterium sp. withfructose-containing substrate can be carried out by contacting theCorynebacterium sp. with fructose which can be the contact of fructosewith at least one selected from the group consisting of a cell mass ofthe strain, a culture of the strain and a lysate of the strain. Inaddition, the reaction of the Corynebacterium sp. withfructose-containing substrate can be carried out by mixing theCorynebacterium sp. with fructose, or by contacting the Corynebacteriumsp. immobilized in the substrate with fructose, so as to convertingfructose to psicose.

For effective production of psicose in the method, fructose, serving asa substrate, is used at a concentration of 40 to 75% (w/v) in thereaction mixture, for example, at a concentration of 50 to 75% (w/v). Alower concentration than the lower limit of fructose may decrease theeconomic feasibility of psicose in this manner. On the other hand, ifpresent at a concentration higher than the upper limit, fructose is lessapt to dissolve. Hence, the concentration preferably falls within therange. Fructose may be in the form of a solution in a buffer or water(e.g., distilled water).

The reaction may be carried out at a pH of 6 to 9.5, for example, at apH of 7 to 9, at a pH of 7 to 8, at a pH of 8 to 9. the reaction may beconducted under the temperature condition of 40° C. or higher, forexample, 4° C. or higher. When the reaction may be conducted at atemperature of 80° C., the substrate fructose may be apt to undergobrowning. Hence, the reaction may be conducted under the temperaturecondition of from 40 to 80° C., for example 50 to 75° C., 60 to 75° C.,or 68 to 75° C.

In addition, a longer period of reaction time leads to a higherconversion rate of psicose. It is recommended to conduct the reactionfor 1 hr or longer, for example, 2 hrs or longer, 3 hrs or longer, 4 hrsor longer, 5 hrs or longer, or 6 hrs or longer. However, the reactiontime is preferably set forth within 48 hrs since when the reaction timeis extended over 48 hrs, the increment of the conversion rate of psicosebecomes slight, or may be decreased. Hence, the reaction time may be setforth to range from 1 to 48 hrs, from 2 to 48 hrs, from 3 to 48 hrs,from 4 to 48 hrs, from 5 to 48 hrs, or from 6 to 48 hrs. Inconsideration of industrial and economic aspects, the reaction time mayfall within the range of 1 to 48 hrs, 2 to 36 hrs, 3 to 24 hrs, 3 to 12hrs, or 3 to 6 hrs, but is not be limited thereto. This condition isselected in order to maximize the conversion yield from fructose topsicose.

In addition, when the recombinant cell is used in the psicose-producingmethod, its concentration may be set forth to range from 5 mg (dcw: drycell weight)/ml or higher in the entire reaction mixture, for example,range from 5 to 100 mg(dcw)/ml, from 10 to 90 mg(dcw)/ml, from 20 to 80mg(dcw)/ml, from 30 to 70 mg(dcw)/ml, from 40 to 60 mg(dcw)/ml, or from45 to 55 mg(dcw)/ml. If the concentration of cell mass is below thelower limit, poor or almost no conversion activity of psicose isexhibited. On the other hand, a concentration exceeding the upper limitmeans crowding of cells which are likely to act as an obstructer to theoptimization of the entire conversion yield of psicose.

The enzymatic protein having psicose conversion activity (for examplepsicose epimerase) may show the property of a metalloenzyme the activityof which is controlled by metal ions. Hence, the presence of a metal ionmay promote the reaction catalyzed by the enzymatic protein, thusincreasing the production yield of psicose.

Therefore, the composition for the production of psicose may furthercomprise a metal ion. the method for producing psicose may furthercomprise adding a metal ion. In one embodiment, the metal ion may beadded to the culture medium in the process of culture, or may be addedduring the culturing process.

In another embodiment, the metal ion may be added to fructose or amixture of fructose and Corynebacterium sp. The metal ion can be addedto a support to which the enzymatic proteins are immobilized (before theaddition of D-fructose) or to a mixture of an enzymaticprotein-immobilized support and D-fructose (after the addition ofD-fructose), or may be added in mixture with D-fructose or individuallytogether with D-fructose.

The metal ion which can contribute to an improvement in the productionyield of psicose may be selected from the group consisting of a copperion, a manganese ion, a calcium ion, a magnesium ion, a zinc ion, anickel ion, a cobalt ion, an iron ion, an aluminum ion, and anycombination thereof. For example, either or both of a manganese ion anda cobalt ion may be used. In consideration of an improvement in theproduction yield of psicose, the metal ion can be added at an amount of0.5 mM or more. when the amount of the metal ion exceeds 5 mM, theeffect of addition is insignificant compared to the surplus amount. So,the amount of the metal ion is set forth to be 5 mM or less. Forexample, the metal ion is used in an amount of 0.5 mM to 5 mM, forexample 0.5 mM to 2 mM.

So long as it establishes an environment for maintaining the activity ofthe strain or the enzymatic protein produced from the strain for a longperiod of time, any support configured to immobilize the strain or theenzymatic protein thereto may be used. For example, sodium alginate mayserve as the support. Sodium alginate, a naturally occurring colloidalpolysaccharide abundantly found in the cell walls of brown algae,consists of β-D-mannuronic acid and α-L-gluronic acid, with a covalent β1-4 linkage therebetween. Allowing for the stable immobilization of thestrain or the enzyme thereto, the linear polymer may be advantageous forthe production yield of psicose.

In one embodiment, a 1.5-4.0% (w/v) sodium alginate solution (e.g.,aqueous sodium alginate solution), for example, an about 2.5% (w/v)sodium alginate solution may be used for immobilizing the strain. By wayof example, a cell mass of the strain, a culture broth containing theenzyme produced by the strain, or a lysate of the strain is mixed with 1to 2 volumes of an aqueous sodium alginate solution, and the mixture isdripped to a 0.2 M calcium ion solution using a syringe pump and avacuum pump, to form beads to which the cell mass of the strain, theculture containing the enzyme produced by the strain, or the lysate ofthe strain are immobilized. The enzyme may be purified from the strain,a culture of the strain or a lysate of the strain using a typicalmethod, for instance, dialysis, precipitation, adsorption,electrophoresis, affinity chromatography, or ion exchangechromatography.

The psicose-producing method comprises the reaction of the enzymaticproteins with D-fructose. In one embodiment, the reaction between theenzymatic proteins and D-fructose may be carried out by contacting theenzymatic proteins with D-fructose.

In one embodiment, the reaction between the enzymatic proteins andfructose may be carried out by contacting the enzymatic proteins withfructose. In another embodiment, the contact between the enzymaticproteins and fructose may be carried out by, for example, mixing theenzymatic proteins with fructose or bringing fructose into contact withthe enzymatic proteins immobilized to a support. In a furtherembodiment, the reaction between the enzymatic proteins and fructose maybe carried out by culturing a cell mass of the recombinant cell in amedium containing fructose. The reaction of the enzymatic proteins withfructose leads to conversion and thus production of psicose fromD-fructose.

In the psicose-producing method, efficiency may be brought in theproduction of psicose when the enzymatic proteins are used at aconcentration of 0.001 mg/ml to 1.0 mg/ml in the reaction mixture, at aconcentration of 0.005 mg/ml to 1.0 mg/ml, at a concentration of 0.01mg/ml to 1.0 mg/ml, at a concentration of 0.01 mg/ml to 0.1 mg/ml, or ata concentration of 0.05 mg/ml to 0.1 mg/ml. When the enzymatic proteinsare used at a concentration lower than the lower limit, the conversionyield of psicose may be poor. On the other hand, too high aconcentration of the enzymatic proteins decreases the industrial economyof psicose production.

For effective production of psicose in the method, fructose, serving asa substrate, is used at a concentration of 40 to 75% (w/v) in thereaction mixture, for example, at a concentration of 50 to 75% (w/v). Alower concentration than the lower limit of fructose may decrease theeconomic feasibility of psicose in this manner. On the other hand, ifpresent at a concentration higher than the upper limit, fructose is lessapt to dissolve. Hence, the concentration preferably falls within therange. Fructose may be in the form of a solution in a buffer or water(e.g., distilled water).

By considering the optimal reaction condition of enzyme protein, thereaction pH, temperature and the enzyme concentration can be adjusted.For example, the reaction pH can be 6 to 9, or the temperature can be30° C. or higher, for example 40° C. or higher, because the fructose maybe apt to undergo browning at 80° C. or higher. In addition, a longerperiod of reaction time leads to a higher conversion rate of psicose. Itis recommended to conduct the reaction for 1 hr or longer, because ofthe heat-stability of enzyme (at 50° C.). When the reaction time exceeds8 hours, it cannot have any significant effect on the conversion rate ofpsicose or can decrease the conversion rate. Thus, the reaction time ispreferable 8 hours or shorter than.

When the recombinant cell is used in the psicose-producing method, itsconcentration may be set forth to range from 5 mg (dcw: dry cellweight)/ml or higher in the entire reaction mixture.

In an embodiment, the method for producing psicose may comprise a stepof reacting the fructose with a recombinant cell expressing psicoseepimerase or the psicose epimerase separated from the recombinant cell.In one embodiment, the method for producing psicose may compriseculturing and recovering a recombinant cell.

After being produced from fructose using the method of the presentinvention, psicose can be purified by a typical method which can bereadily selected by a person skilled in the art, for example, from thegroup consisting of centrifugation, filtration, crystallization, ionexchange chromatography, and a combination.

Advantageous Effects

A gene expression system which expressing the psicose epimerase in alarge amount with GRAS microorganism such as Corynebacterium sp., avector and Corynebacterium sp. are provided according to the presentinvention, and the psicose epimerase obtained by using the geneexpression system can produce psicose form the fructose-containingsubstrate.

BRIEF DESCRIPTION OF DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a cleavage map of a recombinant vector for expressing psicose3-epimerase protein according to one embodiment of the presentinvention.

FIG. 2 is a picture showing the protein amount by using SDS-PAGE oflysate of Corynebacterium sp. cell cultured by using bead beater.

MODE FOR INVENTION

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

Example 1. Plasmid Production Example 1-1: Vector Production with SodPromoter

The nucleotide sequence (CDPE gene; Genbank: EDS06411.1) encodingpsicose epimerase derived from Clostridiuim scindens ATCC 35704 wasoptimized for E. coli to produce a modified nucleotide sequence whichwas called as CDPE. The optimized polynucleotide (SEQ ID NO: 36), sodregulating sequence (SEQ ID NO: 17: sod promoter-RBS-SPACER R1TT-LINKER)derived from Corynebacterium gDNA, and T7 terminator of pET21a vectorwere amplified by PCR method to produce each template and were ligatedto one template according to the overlapping PCR method. The onetemplate was cloned into pGEM T-easy vector according to T-vectorcloning method and was analyzed for the sequence. Specifically, thepolynucleotide included the sod regulating sequence of SEQ ID NO: 17,the optimized CDPE coding sequence for E. coli of SEQ ID NO: 36, andT7-terminator.

The whole polynucleotide was inserted into the same restrictionrecognition site of pCES208 (J. Microbiol. Biotechnol., 18:639-647,2008) with restriction enzyme NotI and XbaI (NEB), to produce therecombinant vector of pCES208/psicose epimerase (pCES_sodCDPE). Thecleavage map of recombinant vector (pCES_sodCDPE) is shown in FIG. 1.

Example 1-2: Vector Production with Saturation Mutagenesis

In order to prepare a vector using the saturation mutagenesis, theprimers including -NN- as a target site were prepared. Specifically, TTin the 3′-end of first RBS(GAAGGA) and the second RBS were decided astarget site and asked Genotec to synthesize the primer. The primersequences, saturation mutagenesis site, and primer binding site weresummarized in Table 2.

TABLE 2 Primer  name sequence(5′ -> 3′) SEQ ID NO RBS1_FGGTGCGGAAACCTACGAAAGGANNTTTTAC 45 CCATGGCTGTATACGAAC RBSl_RGTTCGTATACAGCCATGGGTAAAANNTCCT 46 TTCGTAGGTTTCCGCACC RBS2_FGACTACGCATACGACGAAAGGANNACAAAAT 47 GAAACACGGTATCTACTAC RBS2_RGTAGTAGATACCGTGTTTCATTTTGTNNTCC 48 TTTCGTCGTATGCGTAGTC

The front fragment and the rear fragment divided by a reference site of-NN- were obtained by PCR, to produce one template produced according tothe overlapping PCR method. The template was inserted into pCES208plasmid by ligating with XbaI and NotI site, so as to obtain the plasmidaccording to the Saturation mutagenesis.

Example 2. Transformation and Screening Transformed E. coli Example 2-1:E. coli Transformation

E. coli DH10b strain was transformed with the plasmid obtained inEXAMPLE 1 by electrophoresis and screened. Specifically, kanamycin waspoured to 1.5 ml tube to be 15 μg/ml of the final concentration ofkanamycin, and add with 1 ml of LB (tryptone 10 mg/L, NaCl 10 mg/L,yeast extract 5 mg/L). The randomly-selected colonies were inoculated onthe plate and cultured at 37° C. for about 16 hours. Then, the cell washarvested to remove the culture medium, was reacted with 50% fructose(substrate) dissolved in 50 mM PIPES buffer (pH 7.0) by the addition of1 mM Mn²⁺ at 60° C. for 30 minutes, and quenched at 100° C. for 5minutes.

Example 2-2: Screening with Psicose Conversion Rate

The product of EXAMPLE 2-1 was analyzed with LC analysis to compare theconversion rate of psicose with that of pCES_sodCDPE. Then, thetransformant with modified gene having a higher conversion rate wasselected. Specifically, the conversion rate was obtained by analyzingthe LC peak of substrate (fructose) and product (psicose) and the peakarea.

The comparison of LC peak area confirmed that the decreasing extent ofpsicose production and substrate consumption. The standard curves wereobtained by preparing the samples with different fructose concentrationsof 10, 20, 50, 100, 120, 150 mM and the samples with different psicoseconcentration of 1, 2, 5, 10, 20, 50 mM to be R² of 0.99 or higher.Then, each formula was inferred from the standard curves, and thefructose concentration and psicose concentration were obtained by usingthe LC peak area.

The final values were indicated as psicose conversion rate which wasproportional to the amount of expressed CDPE. Thus, as the amount ofproduced psicose increase, the amount of expressed CDPE increases.

As a result, 6 mutants including three mutants at R1 site and 3 mutantsat R2 site were selected and designated as name R1-1, R1-4, R1-8, R2-1,R2-5, or R2-11. Compared to the LC analysis result of the controlincluding unmodified sequence (pCES_sod CDPE), four mutants wereselected based on the psicose conversion rate and shown in Table 3.

TABLE 3 Sample Psicose conversion rate (%) sod_CDPE 5.15 R1-1 8.59 R1-48.94 R2-5 5.66 R2-11 6.07

As shown in Table 3, finally-selected mutants showing increasedconversion rate of psicose were R1-1 and R1-4 at R1 site and R2-5 andR2-11 at R2 site, and thus, 4 mutants showed increased CDPE expression.

Example 2-3: Identification of Modified Sequence

On the basis of nucleotide sequence of SEQ ID NO: 3 in the unmodifiedpCES_sodCDPE, R1-1 had GA substituted and R1-4 had GG substituted at TTof control target site.

On the basis of nucleotide sequence of SEQ ID NO: 7 in the non-mutatedpCES_sodCDPE, R2-5 and R2-11 had GG substituted at TT of control targetsite.

Example 3. Measurement of CDPE Expression Rate in Corynebacterium

Corynebacterium glutaricum was transformed with the plasmid obtained inEXAMPLE 1 by electrophoresis. The colony was inoculated on LB medium(tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L) enriched withKanamycin to be final concentration of Kanamycin as 15 ug/ml, andcultured at 30° C. and 250 rpm for 16 hours. Then, 1 mL of culturesolution was inoculated in 100 ml LB medium containing 15 ug/ml ofKanamycin and then cultured at 30° C. and 250 rpm for 16 hours.

The recombinant Corynebacterium glutaricum transformed with the plasmidobtained in EXAMPLE 1-1 (pCES_sodCDPE) was deposited on Oct. 29, 2014,at the Korea Culture Center of Microorganisms (KCCM) located at 25Hongjenae-2ga-gil, Seodaemun-gu, Seoul, Republic of Korea, as Accessionnumber of KCCM11593P.

In addition, Corynebacterium glutaricum was transformed with 4 mutantsobtained in EXAMPLE 2 respectively and obtained by culturing them in 100mL of LB medium. The cells were lysed and purified by using His-tagpurification method. Then, the cell lysate was carried out with SDS-PAGEto identify the conversion rate of CDPE.

Specifically, the cultured cells were lysed with Bead beater and thesupernatant was collected, mixed with sample buffer at 1:1 and heated at100° C. for 5 minutes. The prepared sample was analyzed withelectrophoresis by suing 12% SDS-PAGE gel (composition: running gel—3.3ml H₂O, 4.0 ml 30% acrylamide, 2.5 ml 1.5M Tris buffer (pH 8.8), 100 μl10% SDS, 100 μl, 10% APS, 4 μl TEMED/stacking gel—1.4 ml H₂O, 0.33 ml30% acrylamide, 0.25 ml 1.0 M Tris buffer (pH 6.8), 20 μl 10% SDS, 20 μl10% APS, 2 μl TEMED) at 180 V for 50 minutes to identify the proteinexpression.

After identifying the CDPE expression on SDS-PAGE gel, the product waspurified according to His-Tag purification method using Ni-NTA resin,and the conversion rate of psicose was calculated by using the formulaof conversion rate (%)=(Purified protein (mg)/Total soluble protein(mg))*100). The calculated conversion rate was indicated in Table 4.

In following Table 4, the whole cellular proteins means all proteinsinside the cell expressing cell psicose epimerase, and the amount ofpsicose epimerase is referred to an amount of purified psicoseepimerase. Therefore, the conversion rate means the calculated valueshowing a ratio of expressed target protein to the whole cellularproteins.

TABLE 4 sample CDPE conversion rate (%) Sod_CDPE 10 R1-1 15 R1-4 9.5R2-5 8.3 R2-11 8.5

As shown in Table 4, the concentration of purified CDEP of R1-1 showedabout 1.5 times as high as the transformant with recombinant vector(pCES_sodCDPE). On the other hand, other samples showed a low conversionrate.

Example 4. Psicose Production by Using Enzyme Reaction

Corynebacterium glutaricum was transformed with 4 mutants of R1-1, R1-4,R2-5, and R2-11 obtained in EXAMPLE 2 respectively and obtained byculturing them in 100 mL of LB medium. The unpurified crude enzyme wasused for converting 50 mM fructose-containing substrate to psicose.Then, the amount of produced psicose was analyzed.

The mutant cells expressing CDPE were broken. The supernatant includingthe protein was obtained, measured to be 0.007 mg/ml of theconcentration of whole cellular protein, and added to the substratecontaining 50 mM fructose added by 1 mM Mn²⁺. Then, the reaction wascarried out at pH 7.0 PIPES 50 mM and 60° C. for 5, 10, or 15 minutes,and then quenched with heating at 100° C. for 5 minutes.

The conversion rate of psicose was compared by LC analysis.Specifically, the conversion rate was obtained by analyzing the LC peakof substrate (fructose) and product (psicose) and the peak area.

The LC analysis was performed by using Refractive Index Detector(Agilent 1260 RID) of HPLC (Agilent, USA) equipped with Aminex HPX-87Ccolumn (BIO-RAD), water with the temperature of 80° C. as a mobilephase, and the column speed of 0.6 ml/min. Then, the conversion rate ofpsicose was calculated on the basis of the formula of conversion rate byusing the amount of produced psicose and unconsumed fructose measuredfrom the LC peak. The calculated values are shown in Table 5.Conversion rate (%)=Amount of produced psicose (g/l)/(amount of producedpsicose+amount of remaining fructose) (g/l)*100  [Formula]

TABLE 5 Psicose conversion rate (%) Sample at reaction for 5 minutesSod-CDPE 15.66 R1-1 17.24 R1-4 14.39 R2-5 14.10 R2-11 13.65

As shown in Table 5, the conversion rate of R1-1 was higher thansod-CDPEII. Other modified sequence showed a somewhat reduction ofconversion rate, compared to sod-CDPE.

Example 5. Psicose Production by Using Corynebacterium Cell Reaction

Corynebacterium glutaricum was transformed with 4 mutants of R1-1, R1-4,R2-5, and R2-11 obtained in EXAMPLE 2 respectively and obtained byculturing them in 100 mL of LB medium. The substrate containing 50 wt %of fructose was reacted by using the cell reaction and the conversionrate was compared.

Specifically, The 0.5 to 2 mg/ml of mutant cells expressing CDPE wereadded to the substrate containing fructose at solid content of 50 wt %and 1 mM Mn²⁺, reacted at pH 7.0 PIPES 50 mM and 60° C. and quenched byheating at 100° C. for 5 minutes.

The conversion reaction was performed by using each mutant cell and theconversion rate was calculated according to the LC analysis method. theLC analysis was performed by using Refractive Index Detector (Agilent1260 RID) of HPLC (Agilent, USA) equipped with Aminex HPX-87C column(BIO-RAD), water with the temperature of 80° C. as a mobile phase, andthe column speed of 0.6 ml/min. Then, the conversion rate of psicose wascalculated on the basis of the formula of conversion rate by using theamount of produced psicose and unconsumed fructose measured from the LCpeak. The calculated values are shown in Table 6.Conversion rate (%)=amount of produced psicose (g/l)/(amount of producedpsicose+amount of unconsumed fructose) (g/l)*100  [Formula]

TABLE 6 Sample Psicose conversion rate (%) Sod-CDPEII 6.02 R1-1 8.34R1-4 5.99 R2-5 4.79 R2-11 5.29

As shown in Table 6, the conversion rate of mutant R1-1 was higher thansod-CDPEII. Other modified sequence showed a somewhat reduction ofconversion rate, compared to sod-CDPE.

Example 6. Comparison of Heat Stability in Corynebacterium Cell Reaction

Besides the high conversion rate of the cell, the cell converting thepsicose epimerase stably is also important in the industrial field.Therefore, this experiment was carried out to confirm the heat stabilityof the cell.

In order to confirm the heat stability of cell at 50° C., 1.0 mg/ml ofcells pre-treated with surfactant was re-suspended in 50 mM PIPES buffer(pH 7.0) and heated at 50° C. The cell was sampled at each heating hourand was used for the conversion reaction that the sampled cell was addedto substrate containing 50% fructose and 1 mM of Mn²⁺ and reacted at 50°C. for 60 minutes.

The psicose conversion rate and the decreased extent of sampled cellswere shown in Table 7, by referencing zero of conversion rate and zeroof heating time.

TABLE 7 psicose psicose Relative conversion Relative heat conversionheat Reaction rate (%) of stability of rate (%) stability MinutespCES_sodCDPE pCES_sodCDPE of R1-1 of R1-1 0 8.4 100 11.62 100 120 7.589.21 10.77 92.7 240 7.27 86.56 9.56 82.27 360 7.02 83.52 9.03 77.74 5406.81 81.05 9.19 79.1 840 6.54 77.88 8.9 76.6 1020 6.52 77.65 8.4 72.311200 6.15 73.17 7.32 62.98 1320 5.94 70.64 8.04 69.24 1560 5.92 70.428.15 70.14 1680 5.71 67.92 7.75 66.75 1740 5.24 62.32 6.82 58.73

As shown in Table 7, the heat stability of R-1 was not different frompCES_sodCDPE and thus R1-1 mutant had good heat stability. The half-lifeof R1-1 was expected to be about 1800 minutes.

Example 7. Production of Modified Regulating Sequence and CDPEExpression Example 7-1: Vector Production Including a ModifiedRegulating Sequence

TT in the 3′-end of first RBS(GAAGGA) and the second RBS were decided astarget site and asked Genotec to synthesize the -NN-primer in order tosubstitute TT with GT, GC, or GG. The primer sequences, saturationmutagenesis site, and primer binding site were summarized in Table 8.

TABLE 8 Primer sequence(5′ -> 3′) Seq ID No RBS1GT_FGGTGCGGAAACCTACGAAAGGAGTTTTT 49 ACCCATGGCTGTATACGAAC RBS1GT_RGTTCGTATACAGCCATGGGTAAAAACTC 50 CTTTCGTAGGTTTCCGCACC RBS1GC_FGGTGCGGAAACCTACGAAAGGAGCTTTT 51 ACCCATGGCTGTATACGAAC RBS1GC_RGTTCGTATACAGCCATGGGTAAAAGCTC 52 CTTTCGTAGGTTTCCGCACC RBS1GG_FGGTGCGGAAACCTACGAAAGGAGGTTTT 53 ACCCATGGCTGTATACGAAC RBS1GG_RGTTCGTATACAGCCATGGGTAAAACCTC 54 CTTTCGTAGGTTTCCGCACC

The front fragment and the rear fragment divided by a reference site of-NN- were obtained by PCR, to produce one template produced according tothe overlapping PCR method. The template was inserted into pCES208plasmid by ligating with XbaI and NotI site, so as to obtain the plasmidaccording to the Saturation mutagenesis.

Example 7-2: Measurement of CDPE Expression Rate

Corynebacterium glutaricum was transformed with the plasmid includingthe mutated sequence obtained in EXAMPLE 7-2, cultured in 100 ml of LBmedium, and lysed and purified according to the His-tag purificationmethod using Ni-NTA resin. The concentration of whole cellular proteinand the purified protein (CDPE) were measured according to Bradfordassay and the conversion rate of target protein was calculated.

Specifically, the cultured cells were lysed with Bead beater and thesupernatant was collected, mixed with sample buffer at 1:1 and heated at100° C. for 5 minutes. The prepared sample was analyzed withelectrophoresis by suing 12% SDS-PAGE gel (composition: running gel—3.3ml H₂O, 4.0 ml 30% acrylamide, 2.5 ml 1.5M Tris buffer (pH 8.8), 100 μl10% SDS, 100 μl, 10% APS, 4 μl TEMED/stacking gel—1.4 ml H₂O, 0.33 ml30% acrylamide, 0.25 ml 1.0 M Tris buffer (pH 6.8), 20 μl 10% SDS, 20 μl10% APS, 2 μl TEMED) at 180 V for 50 minutes to identify the proteinexpression. FIG. 2 shows a picture showing the protein amount by usingSDS-PAGE of lysate of Corynebacterium sp. cell cultured by using beadbeater.

After identifying the CDPE expression on SDS-PAGE gel, the product waspurified according to His-Tag purification method using Ni-NTA resin,and the conversion rate of psicose was calculated by using the formulaof conversion rate (%)=(Purified protein (mg)/Total soluble protein(mg))*100). The calculated conversion rate was indicated in Table 9.

TABLE 9 Whole cellular psicose epimerase plasmid protein (mg) enzyme(mg) conversion rate (%) pCES_sodCII 10.7 1.1 10.3 R1GA 11.5 1.7 14.8R1GT 10.9 1.6 14.7 R1GC 8.2 0.8 9.8 R1GG 10.8 0.7 6.5

As shown in Table 9, the conversion rates of R1GA and R1 GT were higherthan pCES_sodCDPE. R1 GC shows similar enzyme activity and R1GG showeddecreased enzyme activity.

Example 7-3: Psicose Production with Cellular Reaction

According to the substantially same method of EXAMPLE 5, Corynebacteriumstrain was transformed with mutants respectively, cultured in 100 ml ofLB medium, and add to the psicose conversion reaction to compare thepsicose conversion rate. The result was shown in Table 10.

TABLE 10 sample psicose conversion rate (%) Relative conversion rate (%)R1GG 4.58 100 R1TT 6.73 147 R1GA 9.76 213 R1GT 9.17 200 R1GC 7.39 161

As shown in Table 10, by referencing 100 of psicose conversion rate ofR1GG, the relative conversion rate of R1GA was 213, R1 GT was 200, andR1 GC was 161, and R1 TT was 147. Therefore, all mutant showed increasedconversion rate.

7-4: Comparison of Heat Stability in Cell Reaction

Besides the high conversion rate of the cell, the cell converting thepsicose epimerase stably is also important in the industrial field.Therefore, this experiment was carried out to confirm the heat stabilityof the cell.

In order to confirm the heat stability of cell at 50° C., 1.0 mg/ml ofcells pre-treated with surfactant was re-suspended in 50 mM PIPES buffer(pH 7.0) and heated at 50° C. The cell was sampled at each heating hourand was used for the conversion reaction that the sampled cell was addedto substrate containing 50% fructose and 1 mM of Mn²⁺ and reacted at 50°C. for 60 minutes.

The psicose conversion rate and the decreased extent of sampled cellswere shown in Table 11, by referencing zero of conversion rate and zeroof heating time.

TABLE 11 Reaction minutes pCES_sodCII R1GA R1GT R1GC R1GG 0 100 100 100100 100 240 86.6 82.3 83.9 91 83.8 360 83.5 77.7 74.1 84.8 79.2 840 77.976.6 72.3 84.7 78.1 1200 73.2 75.8 73.2 81.9 64.2 1560 70.4 70.1 66.977.6 62.3 1680 67.9 66.7 65.2 73.8 56.1 1740 62.3 58.7 56.2 52.4 55.3

Example 8. Production of Modified Regulating Sequence and CDPEExpression 8-1: Vector Production Including a Modified RegulatingSequence

As a result of the modified sequence according to the Saturationmutagenesis, TT located in the first spacer after the first RBS affectedthe CDPE expression. Thus, TT located in the first spacer after thefirst RBS was substituted with GT, GC, or GG, tested for the CDPEexpression and selected as R1-1 (GA substituted for TT after the firstRBS). The nucleotide sequence of R1-1 was used as a template forsubstituting TT after the second RBS with GA, GT, GC, or GG. The mutantswere tested for the psicose conversion rate.

The double mutants were produced by using the mutant (R1-1) obtained inEXAMPLE 5 as a template and the following primer in Table 12.

TABLE 12 SEQ ID Primer Sequence (5′ -> 3′) NO RBS1GA/RBS2GA_FGACTACGCATACGACGAAAGGAGAAC 55 AAAATGAAACACGGTATCTACTAC RBS1GA/RBS2GA_RGTAGTAGATACCGTGTTTCATTTTGT 56 TCTCCTTTCGTCGTATGCGTAGTC RBS1GA/RBS2GT_F GACTACGCATACGACGAAAGGAGTAC 57 AAAATGAAACACGGTATCTACTAC RBS1GA/RBS2GT_RGTAGTAGATACCGTGTTTCATTTTGT 58 ACTCCTTTCGTCGTATGCGTAGTC RBS1GA/RBS2GC_FGACTACGCATACGACGAAAGGAGCAC 59 AAAATGAAACACGGTATCTACTAC RBS1GA/RBS2GC_RGTAGTAGATACCGTGTTTCATTTTGT 60 GCTCCTTTCGTCGTATGCGTAGTC RBS1GA/RBS2GG_FGACTACGCATACGACGAAAGGAGGAC 61 AAAATGAAACACGGTATCTACTAC RBS1GA/RBS2GG_RGTAGTAGATACCGTGTTTCATTTTGT 62 CCTCCTTTCGTCGTATGCGTAGTC

8-2: Measurement of CDPE Expression Rate

According to the same method of EXAMPLE 7-2, Corynebacterium glutaricumwas transformed with the plasmid including the modified regulatingsequence. The CDPE conversion rate was determined and indicated in Table13.

As shown in Table 13, the whole cellular proteins means all proteinsinside the cell expressing cell psicose epimerase, and the amount ofpsicose epimerase is referred to an amount of purified psicoseepimerase. Therefore, the conversion rate means the calculated valueshowing a ratio of expressed target protein to the whole cellularproteins.

TABLE 13 whole cellular psicose epimerase conversion rate microorganismprotein (mg) enzyme (mg) (%) pCES_sodCII 10.7 1.1 10.3 R1GA/R2GA 10.31.5 14.5 R1GA/R2GT 10.7 1.6 15.0 R1GA/R2GC 12.8 1.8 14.1 R1GA/R2GG 11.91.7 14.3

As shown in Table 13, the double mutations of R1 GA/R2GA, R1 GA/R2GT,R1GA/R2GC and R1GA/R2GG showed an increased conversion rate of CDPE thanpCES_sodCDPE.

8-3: Psicose Production by Using Cellular Reaction

According to the same method of EXAMPLE 8-2, Corynebacterium glutaricumwas transformed with the plasmid including the modified regulatingsequence, and cultured in 100 ml of LB medium. The CDPE conversion ratewas determined by the cellular reaction and indicated in Table 14.

To identify the product, the conversion rate was obtained by analyzingthe LC peak of substrate (fructose) and product (psicose) and the peakarea. As a result, the initial piscose production rate of cell(Unit/g-DCW) was analyzed by using on various surfactant solutions andindicated in Table 14.

The LC analysis was performed by using Refractive Index Detector(Agilent 1260 RID) of HPLC (Agilent, USA) equipped with Aminex HPX-87Ccolumn (BIO-RAD), water with the temperature of 80° C. as a mobilephase, and the column speed of 0.6 ml/min.

TABLE 14 psicose conversion Sample rate (%) Relative conversion rate (%)R1GG 4.58 100 R1GA/R2GC 9.95 217

As shown in Table 14, the relative conversion rate (%) of doublemutation R1GA/R2GC on showed 217, on the basis of 100 of psicoseconversion rate of R1GG.

8-4: Comparison of Heat Stability in Cellular Reaction

Besides the high conversion rate of the cell, the cell converting thepsicose epimerase stably is also important in the industrial field.Therefore, this experiment was carried out to confirm the heat stabilityof the cell.

In order to confirm the heat stability of cell at 50° C., 1.0 mg/ml ofcells pre-treated with surfactant was re-suspended in 50 mM PIPES buffer(pH 7.0) and heated at 50° C. The cell was sampled at each heating hourand was used for the conversion reaction that the sampled cell was addedto substrate containing 50% fructose and 1 mM of Mn²⁺ and reacted at 50°C. for 60 minutes.

The psicose conversion rate and the decreased extent of sampled cellswere shown in Table 15 by referencing zero of conversion rate and zeroof heating time.

TABLE 15 Reaction times (minutes) pCES_sodCII R1GA/R2GA R1GA/R2GTR1GA/R2GC R1GA/R2GG 0 100 100 100 100 100 240 86.6 86.9 90.8 90.4 88.5360 83.5 90 81.5 92.2 83.6 840 77.9 80.9 68.4 77.4 82.4 1200 73.2 77.364.6 73.6 72.2 1560 70.4 74.1 62.7 70.8 75.4 1680 67.9 70.5 59.6 67.471.8 1740 62.3 66.4 56.9 61.7 63.2

As shown in Table 15, by comparing the heat stability of pCES_sodCDPEand double mutation, the heat stability of double mutation was notdifferent from pCES_sodCDPE and thus the double mutant had good heatstability. Accordingly, the modified regulating sequence affect theexpression of CDPE, but not influence the heat stability.

The invention claimed is:
 1. A gene expression cassette, producing apsicose epimerase in Corynebacterium sp., and comprising: a nucleotidesequence encoding the psicose epimerase; and a regulating sequence beingoperably connected to the nucleotide sequence in the upstream regulatingthe expression of the nucleotide sequence in Corynebacterium sp, andcomprising a promoter, a ribosome binding site (RBS) sequence and afirst spacer sequence in the direction of 5′ to 3′, wherein the promoterincludes the nucleotide sequence of SEQ ID NO: 1, the ribosome bindingsite (RBS) sequence is a nucleotide sequence in a size of 7 to 20 basesincluding the nucleotide sequence of SEQ ID NO: 2, and the first spacersequence is selected from the group consisting of the nucleotidesequences of SEQ ID NO: 3 to SEQ ID NO: 6, wherein the regulatingsequence further comprises a second RBS sequence which is connected to3′-end of the first spacer directly or via a linker sequence in a lengthof 5 to 100 bases, wherein the second RBS sequence is a nucleotidesequence in a size of 7 to 20 bases including the nucleotide sequence ofSEQ ID NO: 2, wherein the regulating sequence further comprises a secondspacer sequence selected from the group consisting of the nucleotidesequences of SEQ ID NO: 7 to SEQ ID NO: 11, wherein the second spacer isconnected to 3′-end of the second RBS.
 2. The gene expression cassetteaccording to claim 1, wherein the linker sequence is a nucleotidesequence in a size of 42 to 100 bp which includes the nucleotidesequence of SEQ ID NO:
 12. 3. The gene expression cassette according toclaim 1, wherein the regulating sequence comprises the promoternucleotide sequence of SEQ ID NO: 1, the RBS nucleotide sequence of SEQID NO: 2, the first spacer sequence selected from the group consistingof the sequences of SEQ ID NO: 3 to SEQ ID NO: 6, the second RBSnucleotide sequence of SEQ ID NO: 2, and the second spacer sequenceselected from the group consisting of the sequences of SEQ ID NO: 7 toSEQ ID NO:
 11. 4. The gene expression cassette according to claim 1,wherein the Corynebacterium sp. is at least one selected from the groupconsisting of Corynebacterium glutamicum, Corynebacteriumacetoglutamicum, Corynebacterium acetoacidophilum, Corynebacteriumthermoaminogenes, Corynebacterium melassecola and Corynebacteriumefficiens.
 5. The gene expression cassette according to claim 1, whereinthe psicose epimerase is derived from Clostridiun scidens, Treponemaprimitia, Ensifer adhaerens or Ruminococcus torques.
 6. The geneexpression cassette according to claim 5, wherein the psicose epimeraseis an amino acid sequence selected from the group consisting ofnucleotides of SEQ ID NO: 33 to SEQ ID NO:
 36. 7. The gene expressioncassette according to claim 6, wherein the nucleotide sequence encodingthe psicose epimerase is a nucleotide sequence selected from the groupconsisting of nucleotides of SEQ ID NO: 37 to SEQ ID NO:
 44. 8. A vectorcomprising an expression cassette of claim
 1. 9. The vector according toclaim 8, wherein the regulating sequence comprises a nucleotide sequenceselected from the group consisting of the sequences of SEQ ID NO: 18 toSEQ ID NO:
 32. 10. The vector according to claim 8, wherein the vectorfurther comprises at least one sequence selected from the groupconsisting of a replication origin, leader sequence, a selection marker,a cloning site, and a restriction enzyme recognition site.
 11. Arecombinant Corynebacterium sp. host cell comprising a gene expressioncassette of claim 1, or being transformed by a gene expression cassetteof claim
 1. 12. The recombinant Corynebacterium sp. host cell accordingto claim 11, wherein the Corynebacterium sp. is at least one selectedfrom the group consisting of Corynebacterium glutamicum, Corynebacteriumacetoglutamicum, Corynebacterium acetoacidophilum, Corynebacteriumthermoaminogenes, Corynebacterium melassecola and Corynebacteriumefficiens.
 13. A composition for producing a psicose epimerase,comprising at least one selected from the group consisting of an psicoseepimerase obtained by using the recombinant Corynebacterium sp., arecombinant cell, a culture of the recombinant cell, a lysate of therecombinant cell and an extract of cell culture or cell lysate, wherethe recombinant Corynebacterium sp. is transformed by a gene expressioncassette of claim 1 or a vector comprising a gene expression cassette ofclaim 1.